The highly specialized cells of the cardiac pacemaking/conduction system (PCS) work together to initiate and synchronize atrial and ventricular contractions. The PCS includes proximal excitatory (nodal) cells and distal conducting, cells including ultimately the Purkinje cells. The excitatory nodal cells initiate excitation and directly cause the atrial cells to contract through rapid spread of depolarization through adjacent cells connected by gap junctions. The distal conducting cells convey the depolarization ultimately to the Purkinje cells, which cause the ventricular cells to contract. Dysfunction of this intricate electrical system results in cardiac arrhythmias or complete heart block and is a source of significant morbidity and mortality (Cheng C F, Kuo H C, Chien K R, Trends Mol Med 9 (2003) 59).
Localized in spatially discrete regions, the excitatory (nodal) cells are specialized non-contractile muscle cells that are able to produce intrinsic excitation in the absence of nervous innervation because they lack a stable resting membrane potential and are thus constantly depolarizing and drifting toward action potential in a spontaneous and rhythmic way. This is called autorhythmicity and these cells pace the heart and are thus termed “pacemaking” cells.
Pacemaking cells are spatially located primarily in the sinoatrial (SA) node, located in the right atrium where the superior vena cava enters the atrium, and secondarily in the atrioventricular (AV) node, located at the fibrous septum between the right atrium and the right ventricle. During a heartbeat, an action potential is generated in cells of the SA node, which have the fastest rate of depolarization. The action potential then spreads to two places, through gap junctions to the neighboring cells of atria, and to the internodal pathways. Internodal pathways are formed of specialized cells that act as a direct pathway for rapid conduction of the action potential to the AV node. These pathways do not use gap junctions to send impulses. The AV node, which has small diameter fibers and fewer gap junctions, delays and controls transmittal of the impulse to the AV or Common Bundle (Bundle of His), thus allowing time for the atria to finish contracting prior to ventricular contraction. The AV Bundle runs from the AV Node through the atrioventricular septum and then splits into the right and left bundle branches that run down the septum between the two ventricles and on through to the Purkinje fibers communicating the impulse to the ventricular muscle. As there are no electrical connections between cardiomyocytes of the atria and the ventricles, the AV Bundle is the only electrical connection between the atria and the ventricles.
Because adequate perfusion is vital to cellular life, cardiac development begins early in embryogenesis. Thus, the murine heart tube forms at approximately day 8 of embryonic development at which time slow, peristaltic contractions occur. This development can be recapitulated in vitro in embryonic stem cells that are allowed to begin differentiation. Embryonic stem (ES) cells are capable of differentiating into any cell type in the body including cells that constitute the specialized PCS in vivo. When murine ES cells are allowed to differentiate as clusters termed embryoid bodies (EBs), rhythmic spontaneous contractions can be observed between 8 to 10 days of differentiation, indicating the presence of cardiomyocytes. ES cells differentiated as EBs have been used to develop numerous model systems for studying cardiomyocyte differentiation because they closely recapitulate developmental gene expression patterns in vitro (Maltsev V A, et al., Circ Res 75 (1994) 233; M van Kempen et al., Cell Physiol Biochem 13 (2003) 263).
A primitive cardiac conduction system that recapitulates what is occurring during embryogenesis is also known to develop in embryonic bodies. The spontaneously contracting regions observed in differentiating EBs contain cells with electrophysiological characteristics of atrial, ventricular, and pacemaking/conducting myocytes (Wobus A M. et al. Ann NY Acad Sci 752 (1995) 460-9). Thus, in addition to the presence of contracting cardiac myocytes, specialized pacemaking and conducting cells are also present in developing liens and cells with “nodal-like” action potentials have been found in single-cell dispersions of EBs (Maltsev V A. et al., Mech Dev 44 (1993) 41). However, without performing electrophysiological experiments there has been no way to know which cells might be specialized pacemaking or conducting cells. While the development of electrical activity has been studied in EBs plated on multi-electrode arrays, this method provides only field potentials in the regions of the surface electrodes and is not always capable of identifying the specific cells that initiate or conduct the action potentials (Banach K, et al., Am J Physiol Hear Circ Physiol 284 (2003) H2114).
Based on the location of their expression in the heart, several genetic markers have been identified with components of the murine cardiac conduction system. However, it has been unclear whether cells expressing these markers actually function as specialized cardiac pacemaking or conducting myocytes (Myers D C & Fishman G I Trends Cardiovasc Med 13 (2003) 289-95; Tamaddon H S, et al., (Circ Res 87 (2000) 929-36; Rentschler S, et al., Development 128 (2001) 1785-92; Gourdie R G, et al., Birth Defects Res Part C Embryo Today 69 (2003) 46-57). Furthermore, because molecular markers of specialized cardiac conducting cells have not been heretofore undefined, it has remained unknown whether the spontaneously contracting regions are simply isolated groups of myocytes or groups of cells in a higher organization.
Several genetic markers have also been identified with components of the developing heart based through targeted disruption of genes and location of expression in the heart. Using a gene targeting knockout/knock-in approach, Kupershmidt S. et al., Circ Res 84 (1999) 146-52, reported that the endogenous expression pattern of minK, which encodes a β-subunit for the cardiac delayed rectifier potassium current (IK), was localized to the central cardiac conduction system in mice and co-localized with connexin 40 in cells of the interventricular bundle branches. Expression of minK has been detected as early as embryonic day 8.25 in mice and continues to be expressed in adults, where it is confined primarily to the more proximal cardiac conduction system from the sinoatrial node through the interventricular bundles (Kondo R P, et al., J Cardiovasc Electrophysiol 14 (2003) 383). Although adult minK null mice are more prone to atrial arrhythmias than wild-type animals, they do not exhibit an overt altered phenotype.
Another marker that has been identified with discrete components of the specialized cardiac conduction system is the proximal 1.5 kb promoter/enhancer region of the chicken GATA6 gene (cGATA6) (Davis D L, et al. Mech Dev 108 (2001) 105-19). The zinc finger transcription factors GATA4-6 are expressed in the developing heart and participate in the activation of a variety of cardiac specific structural genes including α-myosin heavy chain (α-MHC), cardiac troponin-C, atrial natriuretic factor (ANF), brain natriuretic peptide and cardiac troponin-I. Expression of the GATA factors is transcriptionally regulated in a temporal and spatially specific manner through a network of interdependent regulatory events in which other factors interact with target regulatory regions in GATA promoter/enhancers. For example, in the mouse, the homeodomain protein Nkx2.5 binds to a defined region of the GATA6 enhancer and the ensuing regulatory circuit results in development of the cardiac crescent. (reviewed in Molkentin J D, et al., Developmental Biology 217 (2000) 301). Using lacZ expression as a reporter of cGATA6 enhancer activity in transgenic mice, Davis et al. (supra) demonstrated that cGATA6 is expressed in the cardiac primordia, (prior to expression of minK and the formation of the heart tube). Of all the reported markers of the cardiac conduction system, the cGATA6 enhancer exhibits the earliest and most restricted expression pattern (Wessels A, et al., Novartis Found Symp 250 (2003) 44-59; discussion 59-67, 276-9). In the adult mouse, expression becomes restricted to the proximal portion of the specialized cardiac conduction system.
It has emerged that the coordinate expression of discrete genes at specific stages of cardiac development is regulated through the choreographed action of particular sets of transcription factors acting in a sequence specific manner on various cardiac specific enhancers in the untranslated regions of both regulatory transcription factors and structural genes. However, because there are no model systems for studying the cardiac conduction system in vitro, little is known about the molecular identity of the cells that constitute the vital cardiac conduction system. Although several molecular markers have been shown to delineate components of the cardiac conduction system in vivo, the functional characteristics of the individual cells expressing these markers has remained unknown. (Myers D C and Fishman G I, Trends Cardiovasc Med 13 (2003) 289).
Assigning a molecular phenotype to cardiac myocytes from different regions of the heart is difficult. Currently, the most definitive way to characterize a cardiac myocyte is based on electrophysiological properties. Of the known cardiac-specific genes, there is no single gene that identifies a cardiac pacemaking or conducting cell. Most of the characteristics of pacemaking cardiac myocytes have been determined using freshly isolated cells. Both the SA and AV nodes are heterogeneous structures with regard to their cellular content. Part of the difficulty in identifying the cells that function as pacemaking cells in the heterogenous population is due to the lack of well-defined molecular markers.
Thus one of the biggest obstacles to isolating and studying cardiac pacemaking/conducting cells has been the absence of clear molecular markers for these cells that would enable isolation, enrichment and the development of model in vitro systems in which pacemaking cells can discriminated from conducting and contracting cardiomyocytes.
Genetic markers of the cardiac conduction system currently available have been designated as such based on the location of their expression. None of the putative markers of the cardiac conduction system have been shown to identify cells that actually function as specialized cardiac pacemaking or conducting cells and no one has isolated single cells expressing any of these markers to determine if these cells display characteristics of cardiac pacemaking or conducting cells.
What are needed are molecular markers for identification and isolation of the various component cells of the cardiac PCS both for the development of in vitro model systems and for isolation of cells for cardiotransplantation in the treatment of arrhythmia.